DOI 10.1515/bot-2012-0188 Botanica Marina 2013; 56(4): 313–321 Nguyen Xuan Vy*, Laura Holzmeyer and Jutta Papenbrock New record of the seagrass species Halophila major (Zoll.) Miquel in Vietnam: evidence from leaf morphology and ITS analysis Abstract: The seagrass Halophila major (Zoll.) Miquel (i) the number of cross veins, which ranges from 18 to 22, is reported for the first time from Vietnam. It was found and (ii) the ratio of the distance between the intramar- growing with other seagrass species nearshore, 4–6 m ginal vein and the lamina margin at the half-way point deep at Tre Island, Nha Trang Bay. Leaf morphology and along the leaf length, which is 1:20–1:25 (Kuo et al. 2006). phylogenetic analysis based on ribosomal internal tran- Recently, genetic markers, including plastid and nuclear scribed spacer sequences confirmed the identification. sequences, have been used to reveal the genetic relation- There was very little sequence differentiation among sam- ships among members of the genus Halophila. Among the ples of H. major collected in Vietnam and other countries molecular markers used, neither single sequence analysis in the Western Pacific region. A very low evolutionary of the plastid gene encoding the large subunit of ribulose- divergence among H. major populations was found. 1,5-bisphosphate-carboxylase-oxygenase (rbcL) and of the plastid maturase K (matK) nor analysis of the concat- Keywords: Halophila major; internal transcribed spacer; enated sequences of the two plastid markers has resolved new record; seagrass; Vietnam. the two closely related species H. ovalis and H. ovata (Lucas et al. 2012). In contrast, using phylogenetic analy- ses of the nuclear ribosomal internal transcribed spacer *Corresponding author: Nguyen Xuan Vy, Institute of Botany, (ITS1-5.8S-ITS2) region showed that some specimens Leibniz University Hannover, Herrenhäuserstr. 2, D-30419 Hannover, Germany, e-mail: [email protected]; and Institute of identified as H. ovalis belonged to different clades, and Oceanography, Department of Marine Botany, Vietnam Academy of this clearly points to the need for critical taxonomic revi- Science and Technology, 01 Cau Da, Nha Trang City, Vietnam sion of Halophila material across the entire geographical Laura Holzmeyer and Jutta Papenbrock: Institute of Botany, Leibniz range of this genus (Waycott et al. 2002). A reassessment University Hannover, Herrenhäuserstr. 2, D-30419 Hannover, of Halophila species from Japan based on ITS sequences Germany indicated that H. major and H. ovalis are distinct species (Uchimura et al. 2008), which supported investigations by Kuo et al. (2006). Studies of ITS sequences have reported Introduction little or no nucleotide difference between individual Hal- ophila species, such as H. nipponica (Shimada et al. 2012), In Vietnam, two species of Halophila were recorded in H. hawaiiana Doty and B. C. Stone (McDermid et al. 2003), 1885: Halophila ovalis (R. Br.) H. f. and H. beccarii Ascher- and H. stipulacea (Forss.) Ascherson (Ruggiero and Pro- son (Tien 2008). Halophila minor (Zollinger) den Hartog caccini 2004), although the species varied considerably and H. decipiens Ostenf. were added to the list of Halophila in leaf morphology. However, the ITS marker was a useful occurring in Vietnam during a later study (Dai et al. 1998). tool to reveal new records of H. decipiens for regions, such However, the taxonomic diversity of the genus Halophila as the Hawaiian Islands (McDermid et al. 2002) and Kenya has been given little attention until recently. Defining tax- (McMahon and Waycott 2009). ITS sequences also indi- onomic boundaries within the genus Halophila has rep- cated that H. johnsonii Eiseman and H. ovalis were syno- resented a real challenge due to leaf morphological traits nyms (Short et al. 2010). The results of Uchimura et al. that overlap among species (Kuo et al. 2006, Uchimura (2008) based on ITS sequences suggest that H. gaudichau- et al. 2008, Short et al. 2011, Shimada et al. 2012). dii J. Kuo, H. okinawensis J. Kuo, and H. nipponica may be Halophila major (Zoll.) Miquel [formerly known in Japan as conspecific. H. euphlebia Makino but recently classified as H. major by Our initial studies based on morphology and analy- Kuo et al. (2006)] was distinguished from closely related sis of genetic markers (rbcL and matK) indicated that species, such as H. ovalis, H. minor, H. ovata Gaudichaud, H. ovalis collected at Nha Trang Bay showed different and H. nipponica J. Kuo by two main characteristics: traits in comparison with other H. ovalis populations Bereitgestellt von | Technische Informationsbibliothek und Universitaetsbibliothek Hannover TIB/UB Angemeldet Heruntergeladen am | 25.01.16 11:56 314 N.X. Vy et al.: Halophila major – new record for Vietnam based on ITS in Vietnam, although there were no nucleotide differ- Specimens were identified using the keys of den Hartog ences among rbcL sequences and only one different base (1970) and Kuo et al. (2006). pair among matK sequences of the collections (Nguyen Leaves were washed with deionized water to remove et al. 2013). This led to the hypothesis that phylogenetic CTAB buffer completely. Eight to 10 young leaves from one analysis based on ITS sequences would resolve the taxo- individual of each species were homogenized by mortar nomic uncertainties among specimens of Halophila from and pestle in liquid nitrogen, and 100 mg of the finely Nha Trang, and clarify the status of Halophila species powdered plant material was used for DNA extraction. in Vietnam. There have been no previous records of H. DNA extraction was carried out using the Plant Nucle- major in Vietnam (Tien et al. 2002), although it occurs ospin II Kit (Macherey & Nagel, Düren, Germany) follow- in neighboring countries, such as Indonesia and Thai- ing manufacturer’s instructions with slight modifications land (Uchimura et al. 2008). This study documents a new according to Lucas et al. (2012). The region selected for record for H. major in Vietnam. The morphology, location, PCR amplification was the nuclear ITS region including and habitats of H. major are described, and a molecular the 5.8S sequence. Primer pairs used in this study were phylogeny is presented showing the position of H. major P674 5′-CCTTATCATTTAGAGGAAGGAG-3′ (ITS5a) (Stan- from Vietnam in a Western Pacific context. ford et al. 2000) and P675 5′-TCCTCCGCTTATTGATATGC-3′ (ITS4) (White et al. 1990) to amplify a sequence of 700–720 bp consisting of ITS1, 5.8S, and ITS2. The total volume of 25 μl included 1 × Dream Taq Green buffer, 0.2 mm dNTPs, Materials and methods 2 mm MgCl2, 1 U Taq polymerase (MBI Fermentas, St. Leon-Rot, Germany), 10–30 ng template DNA, and 1 pmol Plants of Halophila species (H. beccarii, H. decipiens, of each primer. PCR was performed in a PTC 200 ther- and H. ovalis) were collected from five different loca- mocycler (Biozym-Diagnostik GmbH, Hess Oldendorf, tions: Thi Nai Lagoon, Cu Mong Lagoon, Van Phong Bay, Germany) with a heated lid under the following condi- Nha Trang Bay, and Thuy Trieu Lagoon along the coastal tions: initial denaturation for 4 min at 95°C followed by 30 central provinces in Vietnam (Figure 1) by SCUBA diving cycles of denaturation for 25 s at 95°C, primer annealing or snorkeling in depths of 1–6 m. Sections of plants about for 30 s at 52°C, and extension for 35 s at 72°C, terminated 10–12 cm long in a developmentally comparable state by a final hold at 10°C. All PCR reactions were repeated were collected haphazardly from 10 to 15 different plants, two to four times independently with the same individual which were separated by 10–15 m to avoid collecting from to keep errors (possibly created by the Taq poly merase) the same clone. These plant sections consisting of intact in the final consensus sequence to a minimum. Direct roots, rhizome, and leaves were washed with seawater sequencing of PCR product was done by GATC Biotech in the field to remove epiphytes and debris that were (Konstanz, Germany) from both directions. Consensus attached to the plants. Each plant sample was sorted by sequence was achieved by Clone Manager 9 (Sci-Ed, species, placed in a single plastic bag, kept on ice, and Cary, NC, USA). For comparison, known ITS sequences transferred to the laboratory on the same day. In the labo- of other Halophila species were added to the dataset ratory, samples were rewashed with deionized water to (Table 1). These sequences were aligned by CLUSTAL X remove seawater. Each plant was divided into two parts, (Thompson et al. 1997), and the alignment was further one was pressed as an herbarium voucher specimen and modified by eye. Gaps were considered as missing data. the other was stored in high-salt cetyltrimethylammonium Identical sequences within each species were excluded bromide (CTAB) buffer (Štorchová et al. 2000) for later from the alignment. Additional in-group sequences were DNA extraction and morphological analysis. Herbarium obtained from GenBank (Table 1), and included in the voucher specimens are currently deposited at the Insti- alignment. Halophila angelmannii Ascherson (AF366404) tute of Oceanography, Nha Trang City, Vietnam. Mate- and H. beccarii Ascherson (AF366441) were used as out- rial stored in CTAB buffer was brought to the Institute of group (Waycott et al. 2002). The program jModelTest 0.1.1 Botany, Leibniz University Hannover, Germany, for further (Posada 2008) was used to find the model of sequence analysis. Ten mature leaves were selected from 10 different evolution that fitted the data set best. Phylogenetic analy- plants of each species for morphological measurements, ses were performed using maximum likelihood, neigh- including lamina width, distance from intramarginal vein bor joining (Saitou and Nei 1987) with the model Tamura to lamina margin, cross-vein angle, and the number of 3-parameter + G, maximum parsimony (Felsenstein 1992) cross veins.